Method for rapid in vitro synthesis of bioconjugate vaccines via recombinant production of n-glycosylated proteins in prokaryotic cell lysates

ABSTRACT

Disclosed are methods, systems, components, and compositions for cell-free synthesis of glycosylated proteins. The glycosylated proteins may be utilized in vaccines, including anti-bacterial vaccines. The glycosylated proteins may include a bacterial polysaccharide conjugated to a carrier, which may be utilized to generate an immune response in an immunized host against the polysaccharide conjugated to the carrier. The glycosylated proteins may be synthesized in cell-free glycoprotein synthesis (CFGpS) systems using prokaryote cell lysates that are enriched in components for glycoprotein synthesis such as oligosaccharyltransferases (OSTs) and lipid-linked oligosaccharides (LLOs) including OSTs and LLOs associated with synthesis of bacterial O antigens.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

The present application claims the benefit of priority under 35 U.S.C.§119(e) to U.S. Provisional Patent Application No. 62/362,327, filed onJul. 14, 2016, the content of which is incorporated herein by referencein its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under MCB1413563 awardedby the National Science Foundation. The government has certain rights inthe invention.

BACKGROUND

The field of the invention relates to in vitro synthesis ofN-glycosylated protein in prokaryotic cell lysates. In particular, thefield of the invention relates to the use of N-glycosylated proteinssynthesized in vitro in prokaryotic cell lysates as vaccine conjugatesagainst pathogens such as bacteria.

Conjugate vaccines are among the safest and most effective methods forprevention of life-threatening bacterial infections. Bioconjugatevaccines are a type of conjugate vaccine produced via protein glycancoupling technology (PGCT), in which polysaccharide antigens areconjugated via N-glycosylation to recombinant carrier proteins using abacterial oligosaccharyltransferase (OST) in living Escherichia colicells. Bioconjugate vaccines have the potential to greatly reduce thetime and cost required to produce antibacterial vaccines. However, PGCTis limited by: i) the length of in vivo process development timelines;and ii) the fact that FDA-approved carrier proteins, such as the toxinsfrom Clostridium tetani and Corynebacterium diptheriae, have not yetbeen demonstrated to be compatible with N-linked glycosylation in livingE. coli. Here, we have applied cell-free glycoprotein synthesis (CFGpS)technology to enable rapid in vitro production of bioconjugate vaccinesagainst pathogenic strains of Escherichia coli and Franscicellatularensis in reactions lasting 20 hours. Due to the modular nature ofthe CFGpS system, this cell-free strategy could be easily applied toproduce bioconjugates using FDA-approved carrier proteins or additionalvaccines against pathogenic bacteria whose surface antigen gene clustersare known. We further show that this system can be lyophilized andretain bioconjugate synthesis capability, demonstrating the potentialfor on-demand vaccine production and development in resource-poorsettings. This work represents the first demonstration of bioconjugatevaccine production in E. coli lysates and has promising applications asa portable prototyping or production platform for antibacterial vaccinecandidates.

SUMMARY

Disclosed are methods, systems, components, and compositions forcell-free synthesis of glycosylated proteins. The glycosylated proteinsmay be utilized in vaccines, including anti-bacterial vaccines. Theglycosylated proteins may include a bacterial polysaccharide conjugatedto a carrier, which may be utilized to generate an immune response in animmunized host against the polysaccharide conjugated to the carrier. Theglycosylated proteins may be synthesized in cell-free glycoproteinsynthesis (CFGpS) systems using prokaryote cell lysates that areenriched in components for glycoprotein synthesis such asoligosaccharyltransferases (OSTs) and lipid-linked oligosaccharides(LLOs) including OSTs and LLOs associated with synthesis of bacterialO-antigens. As such, the prokaryote cell lysates may be prepared fromrecombinant prokaryotes that have been engineered to expressheterologous OSTs and/or that have been engineered to expressheterologous glycan synthesis pathways for production of LLOs. Thedisclosed lysates may be described as modular and may be combined toprepare glycosylated proteins in the disclosed CFGpS systems.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Schematic depicting function of C. jejuni N-linked glycosylationpathway expressed in E. coli as adapted from Guarino C., and DeLisa M.P., Glycobiology, 2012 May 22(5):596-601, the content of which isincorporated herein by reference in its entirety.

FIG. 2. Strategies for production of conjugate and bioconjugate vaccinesas adapted from Ihssen et al., Microb. Cell. Fact. 2010 9:61, pages1-13, the content of which is incorporated herein by reference in itsentirety. The schematic illustrates production of an example vaccineagainst Franciscella tularensis.

FIG. 3. Application of CFGpS technology for in vitro production ofbioconjugate vaccines. Example in vivo and in vitro workflows forproduction of anti-F. tularensis bioconjugates. The ability to producebioconjugates in vitro will enable rapid prototyping of novel vaccinecandidates.

FIG. 4. Rapid synthesis of glycoproteins bearing diverse bacterialO-antigens in mixed lysate CFGpS. S30 lysates were prepared from CLM24cells expressing the C. jejuni OST (CjOST lysate), the Franciscellatularensis O-antigen (FtO-PS) biosynthesis pathway (FtLLO lysate), orthe Escherichia coli O78 antigen (EcO78-PS) biosynthesis pathway(EcO78LLO lysate). (A) FtLLO lysate was mixed with CjOST lysate in CFGpSreactions containing DNA template for either sfGFP21-DQNAT-6×His orMBP-4×DQNAT-6×His. The FtO-PS is covalently attached to R4-DQNAT andMBP-4×DQNAT when both the CcOST and FtLLO lysate are present in theCFGpS reaction, indicated by the ladder-like pattern observed in theWestern blot assay (lanes 2, 4). (B) EcO78LLO lysate was mixed withCjOST lysate in CFGpS reactions containing DNA template for eithersfGFP21-DQNAT-6×His, or MBP-4×DQNAT-6×His. The EcO78-PS is covalentlyattached to sfGFP-21-DQNAT and MBP-4×DQNAT when both the CjOST andEcO78LLO lysate are present in the CFGpS reaction, indicated by theladder-like pattern observed in the Western blot assay (lanes 2, 4). Thebioconjugates were also cross-reactive with a commercial antiserumagainst the E. coli O78 strain (data not shown). These resultsdemonstrate the modularity of LLOs in mixed lysate CFGpS and thepotential of CFGpS technology for rapid synthesis of antibacterialvaccines. Abbreviations: CLM24 pSF CjOST; FtLLO lysate: CLM24 pGAB2;α-FtO antigen: FB11 mAb specific for F. tularensis O-antigen glycan.

FIG. 5. Freeze-dried CFGpS reactions for point-of-use vaccinemanufacturing. (A) Scheme for portable bioconjugate production anddistribution in resource-limited settings. (B) Lyophilized CFGpSreactions retain bioconjugate synthesis activity. CFGpS reactions wereprepared containing either CjOST lysate or FtLLO lysate alone, or amixture of both CjOST and FtLLO lysates. These reactions were thenlyophilized and reconstituted with 15 μL nuclease-free water. Pre-mixedreactions (lanes 1, 2, 5, 6) were run directly following reconstitution,while reactions containing CjOST lysate or FtLLO lysate alone were mixedfollowing reconstitution (lanes 3, 4, 7). The FtO-PS is attached to thetarget protein when the DQNAT sequon is synthesized and both CjOSTlysate or FtLLO lysate are present in the reaction (lanes 2, 4, 6, 7).Bioconjugate synthesis capability is preserved following lyophilization,demonstrating the potential of CFGpS for portable bioconjugateproduction and distribution in resource-poor areas. Abbreviations: CjOSTlysate: CLM24 pSF CjOST; FtLLO lysate: CLM24 pGAB2; α-FtO antigen: FB11mAb specific for F. tularensis O-antigen glycan.

FIG. 6. Structures of O-antigens produced in selectively enriched S30lysates. (A) Structure of the F. tularensis Schu S4 O-antigen (B)Structure of the enterotoxigenic E. coli O78 antigen.

DETAILED DESCRIPTION

The present invention is described herein using several definitions, asset forth below and throughout the application.

Definitions

The disclosed subject matter may be further described using definitionsand terminology as follows. The definitions and terminology used hereinare for the purpose of describing particular embodiments only, and arenot intended to be limiting.

As used in this specification and the claims, the singular forms “a,”“an,” and “the” include plural forms unless the context clearly dictatesotherwise. For example, the term “a gene” or “an oligosaccharide” shouldbe interpreted to mean “one or more genes” and “one or moreoligosaccharides,” respectively, unless the context clearly dictatesotherwise. As used herein, the term “plurality” means “two or more.”

As used herein, “about”, “approximately,” “substantially,” and“significantly” will be understood by persons of ordinary skill in theart and will vary to some extent on the context in which they are used.If there are uses of the term which are not clear to persons of ordinaryskill in the art given the context in which it is used, “about” and“approximately” will mean up to plus or minus 10% of the particular termand “substantially” and “significantly” will mean more than plus orminus 10% of the particular term.

As used herein, the terms “include” and “including” have the samemeaning as the terms “comprise” and “comprising.” The terms “comprise”and “comprising” should be interpreted as being “open” transitionalterms that permit the inclusion of additional components further tothose components recited in the claims. The terms “consist” and“consisting of” should be interpreted as being “closed” transitionalterms that do not permit the inclusion of additional components otherthan the components recited in the claims. The term “consistingessentially of” should be interpreted to be partially closed andallowing the inclusion only of additional components that do notfundamentally alter the nature of the claimed subject matter.

The phrase “such as” should be interpreted as “for example, including.”Moreover the use of any and all exemplary language, including but notlimited to “such as”, is intended merely to better illuminate theinvention and does not pose a limitation on the scope of the inventionunless otherwise claimed.

Furthermore, in those instances where a convention analogous to “atleast one of A, B and C, etc.” is used, in general such a constructionis intended in the sense of one having ordinary skill in the art wouldunderstand the convention (e.g., “a system having at least one of A, Band C” would include but not be limited to systems that have A alone, Balone, C alone, A and B together, A and C together, B and C together,and/or A, B, and C together.). It will be further understood by thosewithin the art that virtually any disjunctive word and/or phrasepresenting two or more alternative terms, whether in the description orfigures, should be understood to contemplate the possibilities ofincluding one of the terms, either of the terms, or both terms. Forexample, the phrase “A or B” will be understood to include thepossibilities of “A” or ‘B or “A and B.”

All language such as “up to,” “at least,” “greater than,” “less than,”and the like, include the number recited and refer to ranges which cansubsequently be broken down into ranges and subranges. A range includeseach individual member. Thus, for example, a group having 1-3 membersrefers to groups having 1, 2, or 3 members. Similarly, a group having 6members refers to groups having 1, 2, 3, 4, or 6 members, and so forth.

The modal verb “may” refers to the preferred use or selection of one ormore options or choices among the several described embodiments orfeatures contained within the same. Where no options or choices aredisclosed regarding a particular embodiment or feature contained in thesame, the modal verb “may” refers to an affirmative act regarding how tomake or use and aspect of a described embodiment or feature contained inthe same, or a definitive decision to use a specific skill regarding adescribed embodiment or feature contained in the same. In this lattercontext, the modal verb “may” has the same meaning and connotation asthe auxiliary verb “can.”

As used herein, the terms “bind,” “binding,” “interact,” “interacting,”“occupy” and “occupying” refer to covalent interactions, noncovalentinteractions and steric interactions. A covalent interaction is achemical linkage between two atoms or radicals formed by the sharing ofa pair of electrons (a single bond), two pairs of electrons (a doublebond) or three pairs of electrons (a triple bond). Covalent interactionsare also known in the art as electron pair interactions or electron pairbonds. Noncovalent interactions include, but are not limited to, van derWaals interactions, hydrogen bonds, weak chemical bonds (via short-rangenoncovalent forces), hydrophobic interactions, ionic bonds and the like.A review of noncovalent interactions can be found in Alberts et al., inMolecular Biology of the Cell, 3d edition, Garland Publishing, 1994.Steric interactions are generally understood to include those where thestructure of the compound is such that it is capable of occupying a siteby virtue of its three dimensional structure, as opposed to anyattractive forces between the compound and the site.

Polynucleotides and Synthesis Methods

The terms “nucleic acid” and “oligonucleotide,” as used herein, refer topolydeoxyribonucleotides (containing 2-deoxy-D-ribose),polyribonucleotides (containing D-ribose), and to any other type ofpolynucleotide that is an N glycoside of a purine or pyrimidine base.There is no intended distinction in length between the terms “nucleicacid”, “oligonucleotide” and “polynucleotide”, and these terms will beused interchangeably. These terms refer only to the primary structure ofthe molecule. Thus, these terms include double- and single-stranded DNA,as well as double- and single-stranded RNA. For use in the presentmethods, an oligonucleotide also can comprise nucleotide analogs inwhich the base, sugar, or phosphate backbone is modified as well asnon-purine or non-pyrimidine nucleotide analogs.

Oligonucleotides can be prepared by any suitable method, includingdirect chemical synthesis by a method such as the phosphotriester methodof Narang et al., 1979, Meth. Enzymol. 68:90-99; the phosphodiestermethod of Brown et al., 1979, Meth. Enzymol. 68:109-151; thediethylphosphoramidite method of Beaucage et al., 1981, TetrahedronLetters 22:1859-1862; and the solid support method of U.S. Pat. No.4,458,066, each incorporated herein by reference. A review of synthesismethods of conjugates of oligonucleotides and modified nucleotides isprovided in Goodchild, 1990, Bioconjugate Chemistry 1(3): 165-187,incorporated herein by reference.

The term “amplification reaction” refers to any chemical reaction,including an enzymatic reaction, which results in increased copies of atemplate nucleic acid sequence or results in transcription of a templatenucleic acid. Amplification reactions include reverse transcription, thepolymerase chain reaction (PCR), including Real Time PCR (see U.S. Pat.Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods andApplications (Innis et al., eds, 1990)), and the ligase chain reaction(LCR) (see Barany et al., U.S. Pat. No. 5,494,810). Exemplary“amplification reactions conditions” or “amplification conditions”typically comprise either two or three step cycles. Two-step cycles havea high temperature denaturation step followed by ahybridization/elongation (or ligation) step. Three step cycles comprisea denaturation step followed by a hybridization step followed by aseparate elongation step.

The terms “target,” “target sequence”, “target region”, and “targetnucleic acid,” as used herein, are synonymous and refer to a region orsequence of a nucleic acid which is to be amplified, sequenced, ordetected.

The term “hybridization,” as used herein, refers to the formation of aduplex structure by two single-stranded nucleic acids due tocomplementary base pairing. Hybridization can occur between fullycomplementary nucleic acid strands or between “substantiallycomplementary” nucleic acid strands that contain minor regions ofmismatch. Conditions under which hybridization of fully complementarynucleic acid strands is strongly preferred are referred to as “stringenthybridization conditions” or “sequence-specific hybridizationconditions”. Stable duplexes of substantially complementary sequencescan be achieved under less stringent hybridization conditions; thedegree of mismatch tolerated can be controlled by suitable adjustment ofthe hybridization conditions. Those skilled in the art of nucleic acidtechnology can determine duplex stability empirically considering anumber of variables including, for example, the length and base paircomposition of the oligonucleotides, ionic strength, and incidence ofmismatched base pairs, following the guidance provided by the art (see,e.g., Sambrook et al., 1989, Molecular Cloning—A Laboratory Manual, ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.; Wetmur, 1991,Critical Review in Biochem. and Mol. Biol. 26(3/4):227-259; and Owczarzyet al., 2008, Biochemistry, 47: 5336-5353, which are incorporated hereinby reference).

The term “primer,” as used herein, refers to an oligonucleotide capableof acting as a point of initiation of DNA synthesis under suitableconditions. Such conditions include those in which synthesis of a primerextension product complementary to a nucleic acid strand is induced inthe presence of four different nucleoside triphosphates and an agent forextension (for example, a DNA polymerase or reverse transcriptase) in anappropriate buffer and at a suitable temperature.

A primer is preferably a single-stranded DNA. The appropriate length ofa primer depends on the intended use of the primer but typically rangesfrom about 6 to about 225 nucleotides, including intermediate ranges,such as from 15 to 35 nucleotides, from 18 to 75 nucleotides and from 25to 150 nucleotides. Short primer molecules generally require coolertemperatures to form sufficiently stable hybrid complexes with thetemplate. A primer need not reflect the exact sequence of the templatenucleic acid, but must be sufficiently complementary to hybridize withthe template. The design of suitable primers for the amplification of agiven target sequence is well known in the art and described in theliterature cited herein.

Primers can incorporate additional features which allow for thedetection or immobilization of the primer but do not alter the basicproperty of the primer, that of acting as a point of initiation of DNAsynthesis. For example, primers may contain an additional nucleic acidsequence at the 5′ end which does not hybridize to the target nucleicacid, but which facilitates cloning or detection of the amplifiedproduct, or which enables transcription of RNA (for example, byinclusion of a promoter) or translation of protein (for example, byinclusion of a 5′-UTR, such as an Internal Ribosome Entry Site (IRES) ora 3′-UTR element, such as a poly(A)_(n) sequence, where n is in therange from about 20 to about 200). The region of the primer that issufficiently complementary to the template to hybridize is referred toherein as the hybridizing region.

As used herein, a primer is “specific,” for a target sequence if, whenused in an amplification reaction under sufficiently stringentconditions, the primer hybridizes primarily to the target nucleic acid.Typically, a primer is specific for a target sequence if theprimer-target duplex stability is greater than the stability of a duplexformed between the primer and any other sequence found in the sample.One of skill in the art will recognize that various factors, such assalt conditions as well as base composition of the primer and thelocation of the mismatches, will affect the specificity of the primer,and that routine experimental confirmation of the primer specificitywill be needed in many cases. Hybridization conditions can be chosenunder which the primer can form stable duplexes only with a targetsequence. Thus, the use of target-specific primers under suitablystringent amplification conditions enables the selective amplificationof those target sequences that contain the target primer binding sites.

As used herein, a “polymerase” refers to an enzyme that catalyzes thepolymerization of nucleotides. “DNA polymerase” catalyzes thepolymerization of deoxyribonucleotides. Known DNA polymerases include,for example, Pyrococcus furiosus (Pfu) DNA polymerase, E. coli DNApolymerase I, T7 DNA polymerase and Thermus aquaticus (Taq) DNApolymerase, among others. “RNA polymerase” catalyzes the polymerizationof ribonucleotides. The foregoing examples of DNA polymerases are alsoknown as DNA-dependent DNA polymerases. RNA-dependent DNA polymerasesalso fall within the scope of DNA polymerases. Reverse transcriptase,which includes viral polymerases encoded by retroviruses, is an exampleof an RNA-dependent DNA polymerase. Known examples of RNA polymerase(“RNAP”) include, for example, T3 RNA polymerase, T7 RNA polymerase, SP6RNA polymerase and E. coli RNA polymerase, among others. The foregoingexamples of RNA polymerases are also known as DNA-dependent RNApolymerase. The polymerase activity of any of the above enzymes can bedetermined by means well known in the art.

The term “promoter” refers to a cis-acting DNA sequence that directs RNApolymerase and other trans-acting transcription factors to initiate RNAtranscription from the DNA template that includes the cis-acting DNAsequence.

As used herein, the term “sequence defined biopolymer” refers to abiopolymer having a specific primary sequence. A sequence definedbiopolymer can be equivalent to a genetically-encoded defined biopolymerin cases where a gene encodes the biopolymer having a specific primarysequence.

As used herein, “expression template” refers to a nucleic acid thatserves as substrate for transcribing at least one RNA that can betranslated into a sequence defined biopolymer (e.g., a polypeptide orprotein). Expression templates include nucleic acids composed of DNA orRNA. Suitable sources of DNA for use a nucleic acid for an expressiontemplate include genomic DNA, cDNA and RNA that can be converted intocDNA. Genomic DNA, cDNA and RNA can be from any biological source, suchas a tissue sample, a biopsy, a swab, sputum, a blood sample, a fecalsample, a urine sample, a scraping, among others. The genomic DNA, cDNAand RNA can be from host cell or virus origins and from any species,including extant and extinct organisms. As used herein, “expressiontemplate” and “transcription template” have the same meaning and areused interchangeably.

In certain exemplary embodiments, vectors such as, for example,expression vectors, containing a nucleic acid encoding one or more rRNAsor reporter polypeptides and/or proteins described herein are provided.As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. One type of vector is a “plasmid,” which refers to a circulardouble stranded DNA loop into which additional DNA segments can beligated. Such vectors are referred to herein as “expression vectors.” Ingeneral, expression vectors of utility in recombinant DNA techniques areoften in the form of plasmids. In the present specification, “plasmid”and “vector” can be used interchangeably. However, the disclosed methodsand compositions are intended to include such other forms of expressionvectors, such as viral vectors (e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses), which serveequivalent functions.

In certain exemplary embodiments, the recombinant expression vectorscomprise a nucleic acid sequence (e.g., a nucleic acid sequence encodingone or more rRNAs or reporter polypeptides and/or proteins describedherein) in a form suitable for expression of the nucleic acid sequencein one or more of the methods described herein, which means that therecombinant expression vectors include one or more regulatory sequenceswhich is operatively linked to the nucleic acid sequence to beexpressed. Within a recombinant expression vector, “operably linked” isintended to mean that the nucleotide sequence encoding one or more rRNAsor reporter polypeptides and/or proteins described herein is linked tothe regulatory sequence(s) in a manner which allows for expression ofthe nucleotide sequence (e.g., in an in vitro transcription and/ortranslation system). The term “regulatory sequence” is intended toinclude promoters, enhancers and other expression control elements(e.g., polyadenylation signals). Such regulatory sequences aredescribed, for example, in Goeddel; Gene Expression Technology: Methodsin Enzymology 185, Academic Press, San Diego, Calif. (1990).

Oligonucleotides and polynucleotides may optionally include one or morenon-standard nucleotide(s), nucleotide analog(s) and/or modifiednucleotides. Examples of modified nucleotides include, but are notlimited to diaminopurine, S²T, 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine,5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyl adenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-D46-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,2,6-diaminopurine and the like. Nucleic acid molecules may also bemodified at the base moiety (e.g., at one or more atoms that typicallyare available to form a hydrogen bond with a complementary nucleotideand/or at one or more atoms that are not typically capable of forming ahydrogen bond with a complementary nucleotide), sugar moiety orphosphate backbone.

As utilized herein, a “deletion” means the removal of one or morenucleotides relative to the native polynucleotide sequence. Theengineered strains that are disclosed herein may include a deletion inone or more genes (e.g., a deletion in gmd and/or a deletion in waaL).Preferably, a deletion results in a non-functional gene product. Asutilized herein, an “insertion” means the addition of one or morenucleotides to the native polynucleotide sequence. The engineeredstrains that are disclosed herein may include an insertion in one ormore genes (e.g., an insertion in gmd and/or an insertion in waaL).Preferably, a deletion results in a non-functional gene product. Asutilized herein, a “substitution” means replacement of a nucleotide of anative polynucleotide sequence with a nucleotide that is not native tothe polynucleotide sequence. The engineered strains that are disclosedherein may include a substitution in one or more genes (e.g., asubstitution in gmd and/or a substitution in waaL). Preferably, asubstitution results in a non-functional gene product, for example,where the substitution introduces a premature stop codon (e.g., TAA,TAG, or TGA) in the coding sequence of the gene product. In someembodiments, the engineered strains that are disclosed herein mayinclude two or more substitutions where the substitutions introducemultiple premature stop codons (e.g., TAATAA, TAGTAG, or TGATGA).

In some embodiments, the engineered strains disclosed herein may beengineered to include and express one or heterologous genes. As would beunderstood in the art, a heterologous gene is a gene that is notnaturally present in the engineered strain as the strain occurs innature. A gene that is heterologous to E. coli is a gene that does notoccur in E. coli and may be a gene that occurs naturally in anothermicroorganism (e.g. a gene from C. jejuni) or a gene that does not occurnaturally in any other known microorganism (i.e., an artificial gene).

Peptides, Polypeptides, Proteins, and Synthesis Methods

As used herein, the terms “peptide,” “polypeptide,” and “protein,” referto molecules comprising a chain a polymer of amino acid residues joinedby amide linkages. The term “amino acid residue,” includes but is notlimited to amino acid residues contained in the group consisting ofalanine (Ala or A), cysteine (Cys or C), aspartic acid (Asp or D),glutamic acid (Glu or E), phenylalanine (Phe or F), glycine (Gly or G),histidine (His or H), isoleucine (Ile or I), lysine (Lys or K), leucine(Leu or L), methionine (Met or M), asparagine (Asn or N), proline (Proor P), glutamine (Gln or Q), arginine (Arg or R), serine (Ser or S),threonine (Thr or T), valine (Val or V), tryptophan (Trp or W), andtyrosine (Tyr or Y) residues. The term “amino acid residue” also mayinclude nonstandard or unnatural amino acids. The term “amino acidresidue” may include alpha-, beta-, gamma-, and delta-amino acids.

In some embodiments, the term “amino acid residue” may includenonstandard or unnatural amino acid residues contained in the groupconsisting of homocysteine, 2-Aminoadipic acid, N-Ethyl asparagine,3-Aminoadipic acid, Hydroxylysine, β-alanine, β-Amino-propionic acid,allo-Hydroxylysine acid, 2-Aminobutyric acid, 3-Hydroxyproline,4-Aminobutyric acid, 4-Hydroxyproline, piperidinic acid, 6-Aminocaproicacid, Isodesmosine, 2-Aminoheptanoic acid, allo-Isoleucine,2-Aminoisobutyric acid, N-Methylglycine, sarcosine, 3-Aminoisobutyricacid, N-Methylisoleucine, 2-Aminopimelic acid, 6-N-Methyllysine,2,4-Diaminobutyric acid, N-Methylvaline, Desmosine, Norvaline,2,2′-Diaminopimelic acid, Norleucine, 2,3-Diaminopropionic acid,Ornithine, and N-Ethylglycine. The term “amino acid residue” may includeL isomers or D isomers of any of the aforementioned amino acids.

Other examples of nonstandard or unnatural amino acids include, but arenot limited, to a p-acetyl-L-phenylalanine, a p-iodo-L-phenylalanine, anO-methyl-L-tyrosine, a p-propargyloxyphenylalanine, ap-propargyl-phenylalanine, an L-3-(2-naphthyl)alanine, a3-methyl-phenylalanine, an O-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine,a tri-O-acetyl-GlcNAcpβ-serine, an L-Dopa, a fluorinated phenylalanine,an isopropyl-L-phenylalanine, a p-azido-L-phenylalanine, ap-acyl-L-phenylalanine, a p-benzoyl-L-phenylalanine, an L-phosphoserine,a phosphonoserine, a phosphonotyrosine, a p-bromophenylalanine, ap-amino-L-phenylalanine, an isopropyl-L-phenylalanine, an unnaturalanalogue of a tyrosine amino acid; an unnatural analogue of a glutamineamino acid; an unnatural analogue of a phenylalanine amino acid; anunnatural analogue of a serine amino acid; an unnatural analogue of athreonine amino acid; an unnatural analogue of a methionine amino acid;an unnatural analogue of a leucine amino acid; an unnatural analogue ofa isoleucine amino acid; an alkyl, aryl, acyl, azido, cyano, halo,hydrazine, hydrazide, hydroxyl, alkenyl, alkynl, ether, thiol, sulfonyl,seleno, ester, thioacid, borate, boronate, phosphono, phosphine,heterocyclic, enone, imine, aldehyde, hydroxylamine, keto, or aminosubstituted amino acid, or a combination thereof; an amino acid with aphotoactivatable cross-linker; a spin-labeled amino acid; a fluorescentamino acid; a metal binding amino acid; a metal-containing amino acid; aradioactive amino acid; a photocaged and/or photoisomerizable aminoacid; a biotin or biotin-analogue containing amino acid; a ketocontaining amino acid; an amino acid comprising polyethylene glycol orpolyether; a heavy atom substituted amino acid; a chemically cleavableor photocleavable amino acid; an amino acid with an elongated sidechain; an amino acid containing a toxic group; a sugar substituted aminoacid; a carbon-linked sugar-containing amino acid; a redox-active aminoacid; an α-hydroxy containing acid; an amino thio acid; an α,αdisubstituted amino acid; a β-amino acid; a γ-amino acid, a cyclic aminoacid other than proline or histidine, and an aromatic amino acid otherthan phenylalanine, tyrosine or tryptophan.

As used herein, a “peptide” is defined as a short polymer of aminoacids, of a length typically of 20 or less amino acids, and moretypically of a length of 12 or less amino acids (Garrett & Grisham,Biochemistry, 2^(nd) edition, 1999, Brooks/Cole, 110). In someembodiments, a peptide as contemplated herein may include no more thanabout 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or20 amino acids. A polypeptide, also referred to as a protein, istypically of length ≧100 amino acids (Garrett & Grisham, Biochemistry,2^(nd) edition, 1999, Brooks/Cole, 110). A polypeptide, as contemplatedherein, may comprise, but is not limited to, 100, 101, 102, 103, 104,105, about 110, about 120, about 130, about 140, about 150, about 160,about 170, about 180, about 190, about 200, about 210, about 220, about230, about 240, about 250, about 275, about 300, about 325, about 350,about 375, about 400, about 425, about 450, about 475, about 500, about525, about 550, about 575, about 600, about 625, about 650, about 675,about 700, about 725, about 750, about 775, about 800, about 825, about850, about 875, about 900, about 925, about 950, about 975, about 1000,about 1100, about 1200, about 1300, about 1400, about 1500, about 1750,about 2000, about 2250, about 2500 or more amino acid residues.

A peptide as contemplated herein may be further modified to includenon-amino acid moieties. Modifications may include but are not limitedto acylation (e.g., O-acylation (esters), N-acylation (amides),S-acylation (thioesters)), acetylation (e.g., the addition of an acetylgroup, either at the N-terminus of the protein or at lysine residues),formylation lipoylation (e.g., attachment of a lipoate, a C8 functionalgroup), myristoylation (e.g., attachment of myristate, a C14 saturatedacid), palmitoylation (e.g., attachment of palmitate, a C16 saturatedacid), alkylation (e.g., the addition of an alkyl group, such as anmethyl at a lysine or arginine residue), isoprenylation or prenylation(e.g., the addition of an isoprenoid group such as farnesol orgeranylgeraniol), amidation at C-terminus, glycosylation (e.g., theaddition of a glycosyl group to either asparagine, hydroxylysine,serine, or threonine, resulting in a glycoprotein). Distinct fromglycation, which is regarded as a nonenzymatic attachment of sugars,polysialylation (e.g., the addition of polysialic acid), glypiation(e.g., glycosylphosphatidylinositol (GPI) anchor formation,hydroxylation, iodination (e.g., of thyroid hormones), andphosphorylation (e.g., the addition of a phosphate group, usually toserine, tyrosine, threonine or histidine).

Reference may be made herein to peptides, polypeptides, proteins andvariants thereof. Variants as contemplated herein may have an amino acidsequence that includes conservative amino acid substitutions relative toa reference amino acid sequence. For example, a variant peptide,polypeptide, or protein as contemplated herein may include conservativeamino acid substitutions and/or non-conservative amino acidsubstitutions relative to a reference peptide, polypeptide, or protein.“Conservative amino acid substitutions” are those substitutions that arepredicted to interfere least with the properties of the referencepeptide, polypeptide, or protein, and “non-conservative amino acidsubstitution” are those substitution that are predicted to interferemost with the properties of the reference peptide, polypeptide, orprotein. In other words, conservative amino acid substitutionssubstantially conserve the structure and the function of the referencepeptide, polypeptide, or protein. The following table provides a list ofexemplary conservative amino acid substitutions.

TABLE 1 Original Residue Conservative Substitution Ala Gly, Ser Arg His,Lys Asn Asp, Gln, His Asp Asn, Glu Cys Ala, Ser Gln Asn, Glu, His GluAsp, Gln, His Gly Ala His Asn, Arg, Gln, Glu Ile Leu, Val Leu Ile, ValLys Arg, Gln, Glu Met Leu, Ile Phe His, Met, Leu, Trp, Tyr Ser Cys, ThrThr Ser, Val Trp Phe, Tyr Tyr His, Phe, Trp Val Ile, Leu, Thr

Conservative amino acid substitutions generally maintain: (a) thestructure of the peptide, polypeptide, or protein backbone in the areaof the substitution, for example, as a beta sheet or alpha helicalconformation, (b) the charge or hydrophobicity of the molecule at thesite of the substitution, and/or (c) the bulk of the side chain.Non-conservative amino acid substitutions generally disrupt: (a) thestructure of the peptide, polypeptide, or protein backbone in the areaof the substitution, for example, as a beta sheet or alpha helicalconformation, (b) the charge or hydrophobicity of the molecule at thesite of the substitution, and/or (c) the bulk of the side chain.

Variants comprising deletions relative to a reference amino acidsequence of peptide, polypeptide, or protein are contemplated herein. A“deletion” refers to a change in the amino acid or nucleotide sequencethat results in the absence of one or more amino acid residues ornucleotides relative to a reference sequence. A deletion removes atleast 1, 2, 3, 4, 5, 10, 20, 50, 100, or 200 amino acids residues ornucleotides. A deletion may include an internal deletion or a terminaldeletion (e.g., an N-terminal truncation or a C-terminal truncation of areference polypeptide or a 5′-terminal or 3′-terminal truncation of areference polynucleotide).

Variants comprising fragment of a reference amino acid sequence of apeptide, polypeptide, or protein are contemplated herein. A “fragment”is a portion of an amino acid sequence which is identical in sequence tobut shorter in length than a reference sequence. A fragment may compriseup to the entire length of the reference sequence, minus at least oneamino acid residue. For example, a fragment may comprise from 5 to 1000contiguous amino acid residues of a reference polypeptide, respectively.In some embodiments, a fragment may comprise at least 5, 10, 15, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 150,250, or 500 contiguous amino acid residues of a reference polypeptide.Fragments may be preferentially selected from certain regions of amolecule. The term “at least a fragment” encompasses the full lengthpolypeptide.

Variants comprising insertions or additions relative to a referenceamino acid sequence of a peptide, polypeptide, or protein arecontemplated herein. The words “insertion” and “addition” refer tochanges in an amino acid or sequence resulting in the addition of one ormore amino acid residues. An insertion or addition may refer to 1, 2, 3,4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, or 200 amino acidresidues.

Fusion proteins also are contemplated herein. A “fusion protein” refersto a protein formed by the fusion of at least one peptide, polypeptide,or protein or variant thereof as disclosed herein to at least oneheterologous protein peptide, polypeptide, or protein (or fragment orvariant thereof). The heterologous protein(s) may be fused at theN-terminus, the C-terminus, or both termini of the peptides or variantsthereof.

“Homology” refers to sequence similarity or, interchangeably, sequenceidentity, between two or more polypeptide sequences. Homology, sequencesimilarity, and percentage sequence identity may be determined usingmethods in the art and described herein.

The phrases “percent identity” and “% identity,” as applied topolypeptide sequences, refer to the percentage of residue matchesbetween at least two polypeptide sequences aligned using a standardizedalgorithm. Methods of polypeptide sequence alignment are well-known.Some alignment methods take into account conservative amino acidsubstitutions. Such conservative substitutions, explained in more detailabove, generally preserve the charge and hydrophobicity at the site ofsubstitution, thus preserving the structure (and therefore function) ofthe polypeptide. Percent identity for amino acid sequences may bedetermined as understood in the art. (See, e.g., U.S. Pat. No.7,396,664, which is incorporated herein by reference in its entirety). Asuite of commonly used and freely available sequence comparisonalgorithms is provided by the National Center for BiotechnologyInformation (NCBI) Basic Local Alignment Search Tool (BLAST) (Altschul,S. F. et al. (1990) J. Mol. Biol. 215:403 410), which is available fromseveral sources, including the NCBI, Bethesda, Md., at its website. TheBLAST software suite includes various sequence analysis programsincluding “blastp,” that is used to align a known amino acid sequencewith other amino acids sequences from a variety of databases.

Percent identity may be measured over the length of an entire definedpolypeptide sequence, for example, as defined by a particular SEQ IDnumber, or may be measured over a shorter length, for example, over thelength of a fragment taken from a larger, defined polypeptide sequence,for instance, a fragment of at least 15, at least 20, at least 30, atleast 40, at least 50, at least 70 or at least 150 contiguous residues.Such lengths are exemplary only, and it is understood that any fragmentlength supported by the sequences shown herein, in the tables, figuresor Sequence Listing, may be used to describe a length over whichpercentage identity may be measured.

A “variant” of a particular polypeptide sequence may be defined as apolypeptide sequence having at least 50% sequence identity to theparticular polypeptide sequence over a certain length of one of thepolypeptide sequences using blastp with the “BLAST 2 Sequences” toolavailable at the National Center for Biotechnology Information'swebsite. (See Tatiana A. Tatusova, Thomas L. Madden (1999), “Blast 2sequences—a new tool for comparing protein and nucleotide sequences”,FEMS Microbiol Lett. 174:247-250). Such a pair of polypeptides may show,for example, at least 60%, at least 70%, at least 80%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% or greatersequence identity over a certain defined length of one of thepolypeptides. A “variant” may have substantially the same functionalactivity as a reference polypeptide (e.g., glycosylase activity or otheractivity). “Substantially isolated or purified” amino acid sequences arecontemplated herein. The term “substantially isolated or purified”refers to amino acid sequences that are removed from their naturalenvironment, and are at least 60% free, preferably at least 75% free,and more preferably at least 90% free, even more preferably at least 95%free from other components with which they are naturally associated.

As used herein, “translation template” refers to an RNA product oftranscription from an expression template that can be used by ribosomesto synthesize polypeptides or proteins.

The term “reaction mixture,” as used herein, refers to a solutioncontaining reagents necessary to carry out a given reaction. A reactionmixture is referred to as complete if it contains all reagents necessaryto perform the reaction. Components for a reaction mixture may be storedseparately in separate container, each containing one or more of thetotal components. Components may be packaged separately forcommercialization and useful commercial kits may contain one or more ofthe reaction components for a reaction mixture.

The steps of the methods described herein can be performed in anysuitable order unless otherwise indicated herein or otherwise clearlycontradicted by context. The steps may be repeated or reiterated anynumber of times to achieve a desired goal unless otherwise indicatedherein or otherwise clearly contradicted by context.

Preferred aspects of this invention are described herein, including thebest mode known to the inventors for carrying out the invention.Variations of those preferred aspects may become apparent to those ofordinary skill in the art upon reading the foregoing description. Theinventors expect a person having ordinary skill in the art to employsuch variations as appropriate, and the inventors intend for theinvention to be practiced otherwise than as specifically describedherein. Accordingly, this invention includes all modifications andequivalents of the subject matter recited in the claims appended heretoas permitted by applicable law. Moreover, any combination of theabove-described elements in all possible variations thereof isencompassed by the invention unless otherwise indicated herein orotherwise clearly contradicted by context.

Cell-Free Protein Synthesis (CFPS)

The strains and systems disclosed herein may be applied to cell-freeprotein synthesis methods as known in the art. See, for example, U.S.Pat. Nos. 4,496,538; 4,727,136; 5,478,730; 5,556,769; 5,623,057;5,665,563; 5,679,352; 6,168,931; 6,248,334; 6,531,131; 6,869,774;6,994,986; 7,118,883; 7,189,528; 7,338,789; 7,387,884; 7,399,610;8,703,471; and 8,999,668. See also U.S. Published Application Nos.2015-0259757, 2014-0295492, 2014-0255987, 2014-0045267, 2012-0171720,2008-0138857, 2007-0154983, 2005-0054044, and 2004-0209321. See also U.SPublished Application Nos. 2005-0170452; 2006-0211085; 2006-0234345;2006-0252672; 2006-0257399; 2006-0286637; 2007-0026485; 2007-0178551.See also Published PCT International Application Nos. 2003/056914;2004/013151; 2004/035605; 2006/102652; 2006/119987; and 2007/120932. Seealso Jewett, M. C., Hong, S. H., Kwon, Y. C., Martin, R. W., and DesSoye, B. J. 2014, “Methods for improved in vitro protein synthesis withproteins containing non standard amino acids,” U.S. Patent ApplicationSer. No. 62/044,221; Jewett, M. C., Hodgman, C. E., and Gan, R. 2013,“Methods for yeast cell-free protein synthesis,” U.S. Patent ApplicationSer. No. 61/792,290; Jewett, M. C., J. A. Schoborg, and C. E. Hodgman.2014, “Substrate Replenishment and Byproduct Removal Improve YeastCell-Free Protein Synthesis,” U.S. Patent Application Ser. No.61/953,275; and Jewett, M. C., Anderson, M. J., Stark, J. C., Hodgman,C. E. 2015, “Methods for activating natural energy metabolism forimproved yeast cell-free protein synthesis,” U.S. Patent ApplicationSer. No. 62/098,578. See also Guarino, C., & DeLisa, M. P. (2012). Aprokaryote-based cell-free translation system that efficientlysynthesizes glycoproteins. Glycobiology, 22(5), 596-601. The contents ofall of these references are incorporated in the present application byreference in their entireties.

In certain exemplary embodiments, one or more of the methods describedherein are performed in a vessel, e.g., a single, vessel. The term“vessel,” as used herein, refers to any container suitable for holdingon or more of the reactants (e.g., for use in one or more transcription,translation, and/or glycosylation steps) described herein. Examples ofvessels include, but are not limited to, a microtitre plate, a testtube, a microfuge tube, a beaker, a flask, a multi-well plate, acuvette, a flow system, a microfiber, a microscope slide and the like.

In certain exemplary embodiments, physiologically compatible (but notnecessarily natural) ions and buffers are utilized for transcription,translation, and/or glycosylation, e.g., potassium glutamate, ammoniumchloride and the like. Physiological cytoplasmic salt conditions arewell-known to those of skill in the art.

The strains and systems disclosed herein may be applied to cell-freeprotein methods in order to prepare glycosylated macromolecules (e.g.,glycosylated peptides, glycosylated proteins, and glycosylated lipids).Glycosylated proteins that may be prepared using the disclosed strainsand systems may include proteins having N-linked glycosylation (i.e.,glycans attached to nitrogen of asparagine and/or arginine side-chains)and/or O-linked glycosylation (i.e., glycans attached to the hydroxyloxygen of serine, threonine, tyrosine, hydroxylysine, and/orhydroxyproline). Glycosylated lipids may include O-linked glycans via anoxygen atom, such as ceramide.

The glycosylated macromolecules disclosed herein may include unbranchedand/or branched sugar chains composed of monomers as known in the artsuch as, but not limited to, glucose (e.g., β-D-glucose), galactose(e.g., β-D-galactose), mannose (e.g., β-D-mannose), fucose (e.g.,α-L-fucose), N-acetyl-glucosamine (GlcNAc), N-acetyl-galactosamine(GalNAc), neuraminic acid, N-acetylneuraminic acid (i.e., sialic acid),and xylose, which may be attached to the glycosylated macromolecule,growing glycan chain, or donor molecule (e.g., a donor lipid and/or adonor nucleotide) via respective glycosyltransferases (e.g.,oligosaccharyltransferases, GlcNAc transferases, GalNAc transferases,galactosyltransferases, and sialyltransferases). The glycosylatedmacromolecules disclosed herein may include glycans as known in the art.

The disclosed cell-free protein synthesis systems may utilize componentsthat are crude and/or that are at least partially isolated and/orpurified. As used herein, the term “crude” may mean components obtainedby disrupting and lysing cells and, at best, minimally purifying thecrude components from the disrupted and lysed cells, for example bycentrifuging the disrupted and lysed cells and collecting the crudecomponents from the supernatant and/or pellet after centrifugation. Theterm “isolated or purified” refers to components that are removed fromtheir natural environment, and are at least 60% free, preferably atleast 75% free, and more preferably at least 90% free, even morepreferably at least 95% free from other components with which they arenaturally associated.

Cell-Free Glycoprotein Synthesis (CFGpS) in Prokaryotic Cell LysatesEnriched with Components for Glycosylation

Disclosed are compositions and methods for performing cell-freeglycoprotein synthesis (CFGpS). In some embodiments, the composition andmethods include or utilize prokaryotic cell lysates enriched withcomponents for glycosylation and prepared from genetically modifiedstrains of prokaryotes.

In some embodiments, the genetically modified prokaryote is agenetically modified strain of Escherichia coli or any other prokaryotesuitable for preparing a lysate for CFGpS. Optionally, the modifiedstrain of Escherichia coli is derived from rEc.C321. Preferably, themodified strain includes genomic modifications (e.g., deletions of genesrendering the genes inoperable) that preferably result in lysatescapable of high-yielding cell-free protein synthesis. Also, preferably,the modified strain includes genomic modification (e.g., deletions ofgenes rendering the genes inoperable) that preferably result in lysatescomprising sugar precursors for glycosylation at relatively highconcentrations (e.g., in comparison to a strain not having the genomicmodification). In some embodiments, a lysate prepared from the modifiedstrain comprises sugar precursors at a concentration that is at least20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, or higher thana lysate prepared from a strain that is not modified.

In some embodiments, the modified strain includes a modification thatresults in an increase in the concentration of a monosaccharide utilizedin glycosylation (e.g., glucose, mannose, N-acetyl-glucosamine (GlcNAc),N-acetyl-galactosamine (GalNAc), galactose, sialic acid, neuraminicacid, fucose). As such, the modification may inactivate an enzyme thatmetabolizes a monosaccharide or polysaccharide utilized inglycosylation. In some embodiments, the modification inactivates adehydratase or carbon-oxygen lyase enzyme (EC 4.2) (e.g., via a deletionof at least a portion of the gene encoding the enzyme). In particular,the modification may inactivate a GDP-mannose 4,6-dehydratase (EC4.2.1.47). When the modified strain is E. coli, the modification mayinclude an inactivating modification in the gmd gene (e.g., via adeletion of at least a portion of the gmd gene). The sequence of the E.coli gmd gene is provided herein as SEQ ID NO:1 and the amino acidsequence of E. coli GDP-mannose 4,6-dehydratase is provided as SEQ IDNO:2.

In some embodiments, the modified strain includes a modification thatinactivates an enzyme that is utilized in the glycosyltransferasepathway. In some embodiments, the modification inactivates anoligosaccharide ligase enzyme (e.g., via a deletion of at least aportion of the gene encoding the enzyme). In particular, themodification may inactivate an O-antigen ligase that optionallyconjugates an O-antigen to a lipid A core oligosaccharide. Themodification may include an inactivating modification in the waaL gene(e.g., via a deletion of at least a portion of the waaL gene). Thesequence of the E. coli waaL gene is provided herein as SEQ ID NO:3 andthe amino acid sequence of E. coli O-antigen ligase is provided as SEQID NO:4.

In some embodiments, the modified strain includes a modification thatinactivates a dehydratase or carbon-oxygen lyase enzyme (e.g., via adeletion of at least a portion of the gene encoding the enzyme) and alsothe modified strain includes a modification that inactivates anoligosaccharide ligase enzyme (e.g., via a deletion of at least aportion of the gene encoding the enzyme). The modified strain mayinclude an inactivation or deletion of both gmd and waaL.

In some embodiments, the modified strain may be modified to express oneor more orthogonal or heterologous genes. In particular, the modifiedstrain may be genetically modified to express an orthogonal orheterologous gene that is associated with glycoprotein synthesis such asa glycosyltransferase (GT) which is involved in the lipid-linkedoligosaccharide (LLO) pathway. In some embodiments, the modified strainmay be modified to express an orthogonal or heterologousoligosaccharyltransferase (EC 2.4.1.119) (OST).Oligosaccharyltransferases or OSTs are enzymes that transferoligosaccharides from lipids to proteins.

In particular, the modified strain may be genetically modified toexpress an orthogonal or heterologous gene in a glycosylation system(e.g., an N-linked glycosylation system and/or an O-linked glycosylationsystem). The N-linked glycosylation system of Campylobacter jejuni hasbeen transferred to E. coli. (See Wacker et al., “N-linked glycosylationin Campylobacter jejuni and its functional transfer into E. coli,”Science 2002, Nov. 29; 298(5599):1790-3, the content of which isincorporated herein by reference in its entirety). In particular, themodified strain may be modified to express one or more genes of the pgllocus of C. jejuni or one or more genes of a homologous pgl locus. Thegenes of the pgl locus include pglG, pglF, pglE, wlaJ, pglD, pglC, pglA,pglB, pglJ, pglI, pglH, pglK, and gne, and are used to synthesizelipid-linked oligosaccharides (LLOs) and transfer the oligosaccharidemoieties of the LLOs to a protein via an oligosaccharyltransferase.

Suitable orthogonal or heterologous oligosaccharyltransferases (OST)which may be expressed in the genetically modified strains may includeCampylobacter jejuni oligosaccharyltransferase PglB. The gene for the C.jejuni OST is referred to as pglB, which sequence is provided as SEQ IDNO:5 and the amino acid sequence of C. jejuni PglB is provided as SEQ IDNO:6. PglB catalyzes transfer of an oligosaccharide to a D/E-Y-N-X-S/Tmotif (Y, X≠P) present on a protein.

Crude cell lysates may be prepared from the modified strains disclosedherein. The crude cell lysates may be prepared from different modifiedstrains as disclosed herein and the crude cell lysates may be combinedto prepare a mixed crude cell lysate. In some embodiments, one or morecrude cell lysates may be prepared from one or more modified strainsincluding a genomic modification (e.g., deletions of genes rendering thegenes inoperable) that preferably result in lysates comprising sugarprecursors for glycosylation at relatively high concentrations (e.g., incomparison to a strain not having the genomic modification). In someembodiments, one or more crude cell lysates may be prepared from one ormore modified strains that have been modified to express one or moreorthogonal or heterologous genes or gene clusters that are associatedwith glycoprotein synthesis. Preferably, the crude cell lysates or mixedcrude cell lysates are enriched in glycosylation components, such aslipid-linked oligosaccharides (LLOs), glycosyltransferases (GTs),oligosaccharyltransferases (OSTs), or any combination thereof. Morepreferably, the crude cell lysates or mixed crude cell lysates areenriched in Man₃GlcNAc₂ LLOs representing the core eukaryotic glycanand/or Man₃GlcNAc₄Gal₂Neu₅Ac₂ LLOs representing the fully sialylatedhuman glycan.

The disclosed crude cell lysates may be used in cell-free glycoproteinsynthesis (CFGpS) systems to synthesize a variety of glycoproteins. Theglycoproteins synthesized in the CFGpS systems may include prokaryoticglycoproteins and eukaryotic proteins, including human proteins. TheCFGpS systems may be utilized in methods for synthesizing glycoproteinsin vitro by performing the following steps using the crude cell lysatesor mixtures of crude cell lysates disclosed herein: (a) performingcell-free transcription of a gene for a target glycoprotein; (b)performing cell-free translation; and (c) performing cell-freeglycosylation. The methods may be performed in a single vessel ormultiple vessels. Preferably, the steps of the synthesis method may beperformed using a single reaction vessel. The disclosed methods may beused to synthesis a variety of glycoproteins, including prokaryoticglycoproteins and eukaryotic glycoproteins.

Bioconjugate Vaccine Production

While protein-glycan coupling technology (PGCT) represents a simplifiedand cost-effective strategy for bioconjugate vaccine production, it hasthree main limitations. First, process development timelines,glycosylation pathway design-build-test (DBT) cycles, and bioconjugateproduction are all limited by cellular growth. Second, it has not yetbeen shown whether FDA-approved carrier proteins, such as the toxinsfrom Clostridium tetani and Corynebacterium diptheriae, have not yetbeen demonstrated to be compatible with N-linked glycosylation in livingE. coli. Third, select non-native glycans are known to be transferredwith low efficiency by the C. jejuni oligosaccharyltransferase (OST),PglB.

A modular, in vitro platform for production of bioconjugates has thepotential to address all of these limitations. Here, we demonstrate thatbioconjugates against pathogenic strains of Franciscella tualrensis andEscherichia coli can be produced through coordinated in vitrotranscription, translation, and N-glycosylation in cell-freeglycoprotein synthesis (CFGpS) reactions lasting just 20 hours. Thissystem has the potential to reduce process development and distributiontimelines for novel antibacterial vaccines from weeks to days. Further,because of the modular nature of the CFGpS platform and the fact thatcell-free systems have demonstrated advantages for production ofmembrane proteins compared to living cells, this method could be readilyapplied to produce bioconjugates using FDA-approved carrier proteins,such as the Clostridium tetani and Corynebacterium diptheriae toxins,which are membrane localized. This could be accomplished simply bysupplying plasmid encoding these carrier proteins to CFGpS reactions.Additionally, because of the modular nature of CFGpS, the in vitroapproach could be used to prototype other natural or engineered homologsof the archetypal C. jejuni OST to identify candidate OSTs with improvedefficiency for transfer of O-antigen LLOs of interest. This can beaccomplished by enriching lysates with OSTs of interest and mixing themwith LLO lysates in mixed lysate CFGpS reactions, as we have describedpreviously. (See WO 2017/117539, the content of which is incorporatedherein by reference in its entirety). Finally, we demonstrate thatcell-free bioconjugate synthesis reactions can be lyophilized and retainbioconjugate synthesis capability, demonstrating the potential of theCFGpS system for on-demand, portable, and low cost production ordevelopment efforts for novel vaccines. This novel method for in vitrobioconjugate vaccine production has demonstrated advantages for rapid,modular, and portable vaccine prototyping and production compared toexisting methods. Our technology enables rapid production ofbioconjugate vaccines directed against a user-specified bacterial targetfor therapeutic development or fundamental research.

The present inventors are not aware of any prokaryotic cell-free systemwith the capability to produce glycoproteins or bioconjugate vaccines.There are commercial eukaryotic cell lysate systems for cell-freeglycoprotein production (Promega, ThermoFisher), but these systems donot involve overexpression of orthogonal glycosylation machinery and donot enable modular, user-specified glycosylation in the way our systemcan. For this reason, we feel that there is still substantial commercialpromise for our invention.

The presently disclosed method for in vitro bioconjugate vaccineproduction has demonstrated advantages for rapid, modular, and portablevaccine prototyping and production compared to existing methods. Thissystem addresses limitations of existing production approaches, makingit an attractive alternative or complementary strategy for antibacterialvaccine production. In light of the growing healthcare concerns causedby antibiotic-resistant bacterial infections, this method has thepotential to be extremely valuable for development, production, anddistribution of novel vaccines against diverse pathogenic bacterialstrains. Advantages of the disclosed method include: on demandexpression of bioconjugate vaccines; prototyping novel bioconjugatevaccine candidates; prototyping novel bioconjugate vaccine productionpathways; distribution of bioconjugate vaccines to resource-poorsettings.

In summary, we disclose the first prokaryotic cell-free system capableof coordinated, cell-free transcription, translation, and glycosylationof glycoprotein vaccines. The disclosed system enables production ofbioconjugate vaccines in 20 hours. The modularity of the system enablesrapid prototyping of novel glycosylation pathways and vaccine candidateswith various carrier proteins. The components and products of the systemmaybe lyophilized, which enables the potential for broad and rapiddistribution of vaccine production technology. The disclosed systemreduces the time required to produce bioconjugates in a prokaryotic celllysate from weeks to days, which could provide competitive advantage incommercialization of the technology.

The disclosed bioconjugates may be formulated as vaccines whichoptionally may include additional agents for inducing and/orpotentiating an immune response such as adjuvants. The term “adjuvant”refers to a compound or mixture that enhances an immune response. Anadjuvant can serve as a tissue depot that slowly releases the antigenand also as a lymphoid system activator that non-specifically enhancesthe immune response. Examples of adjuvants which may be utilized in thedisclosed compositions include but are not limited to, co-polymeradjuvants (e.g., Pluronic L121® brand poloxamer 401, CRL1005, or a lowmolecular weight co-polymer adjuvant such as Polygen® adjuvant), poly(I:C), R-848 (a Th1-like adjuvant), resiquimod, imiquimod, PAM3CYS,aluminum phosphates (e.g., AlPO₄), loxoribine, potentially useful humanadjuvants such as BCG (Bacille Calmette-Guerin) and Corynebacteriumparvum, CpG oligodeoxynucleotides (ODN), cholera toxin derived antigens(e.g., CTA1-DD), lipopolysaccharide adjuvants, complete Freund'sadjuvant, incomplete Freund's adjuvant, saponin (e.g., Quil-A), mineralgels such as aluminum hydroxide, surface active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil or hydrocarbonemulsions in water (e.g., MF59 available from Novartis Vaccines orMontanide ISA 720), keyhole limpet hemocyanins, and dinitrophenol.

ILLUSTRATIVE EMBODIMENTS

The following embodiments are illustrative and are not intended to limitthe scope of the claimed subject matter.

Embodiment 1

A method for synthesizing a N-glycosylated recombinant protein carrierwhich may be utilized as a bioconjugate vaccine, the method comprisingperforming coordinated transcription, translation, and N-glycosylationof the recombinant protein carrier thereby providing the N-glycosylatedrecombinant protein carrier which may be utilized as the bioconjugatevaccine, wherein the N-glycosylated recombinant protein carriercomprises: (i) a consensus sequence (which optionally is inserted in theprotein carrier), N-X-S/T, wherein X may be any natural or unnaturalamino acid except proline; and (ii) at least one antigenicpolysaccharide from at least one bacterium N-linked to the recombinantprotein carrier, wherein the at least one antigenic polysaccharideoptionally is at least one bacterial O-antigen, optionally from one ormore strains of E. coli or Franciscella tularensis; and the bioconjugatevaccine optionally may include an adj uvant.

Embodiment 2

The method of embodiment 1, wherein the carrier protein is an engineeredvariant of E. coli maltose binding protein (MBP).

Embodiment 3

The method of embodiment 1, wherein the carrier protein is a detoxifiedvariant of the toxin from Clostridium tetani.

Embodiment 4

The method of embodiment 1, wherein the carrier protein is a detoxifiedvariant of the toxin from Corynebacterium diptheriae.

Embodiment 5

The method of any of the foregoing embodiments, wherein the methodutilizes an oligosaccharyltransferase (OST) which is a naturallyoccurring bacterial homolog of C. jejuni PglB.

Embodiment 6

The method of any of embodiments 1-4, wherein the method utilizes an OSTthat is an engineered variant of C. jejuni PglB.

Embodiment 7

The method of any of embodiments 1-4, wherein the method utilizes an OSTthat is a naturally occurring archaeal OST.

Embodiment 8

The method of any of embodiments 1-4, wherein the method utilizes an OSTwhich is a naturally occurring single-subunit eukaryotic OST, such asthose found in Trypanosoma bruceii.

Embodiment 9

A method for crude cell lysate preparation in which orthogonal genes orgene clusters are expressed in a source strain for the crude celllysate, which results in lysates enriched with glycosylation components(lipid-linked oligosaccharides (LLOs), oligosaccharyltransferases(OSTs), and/or both LLOs and OSTs), and optionally which results in aseparate lysate enriched with LLOs (e.g., LLOs associated withO-antigen) and a separate lysate enriched with OSTs (e.g., for which theLLOs are a substrate), and optionally combining the separate lysates toperform cell-free protein synthesis of a carrier protein which isglycosylated with the glycan component of the LLOs via the OST's enzymeactivity, and further optionally purifying the glycosylated carrierprotein and optionally administering the glycosylated carrier protein asan immunogen.

Embodiment 10

The method of embodiment 9, in which the source strain overexpresses agene encoding an oligosaccharyltransferase (OST).

Embodiment 11

The method of embodiment 9 or 10, in which the source strainoverexpresses a synthetic glycosyltransferase pathway, resulting in theproduction of O-antigens, optionally O-antigens from F. tularensis SchuS4 lipid-linked oligosaccharides (FtLLOs).

Embodiment 12

The method of embodiment 9 or 10, in which the source strainoverexpresses a synthetic glycosyltransferase pathway, resulting in theproduction of O-antigens, optionally O-antigens from enterotoxigenic E.coli O78 lipid-linked oligosaccharides (EcO78LLOs).

Embodiment 13

The method of any of embodiments 9-12, in which the source strainoverexpresses a glycosyltransferase pathway and an OST, resulting in theproduction of LLOs and OST.

Embodiment 14

The method of embodiment 9 or 10, in which the source strainoverexpresses an O-antigen glycosyltransferase pathway from a pathogenicbacterial strain, resulting in the production of O-antigen lipid-linkedoligosaccharides (LLOs).

Embodiment 15

A method for cell-free production of a bioconjugate vaccine thatinvolves mixing crude cell lysates (e.g., any of the crude cell lysatesof embodiments 9-14).

Embodiment 16

The method of embodiment 15, in which the bioconjugate vaccine comprisesan immunogenic carrier that is a protein.

Embodiment 17

The method of embodiment 15, in which the bioconjugate vaccine comprisesan immunogenic carrier that is a peptide.

Embodiment 18

The method of any of embodiments 1-17 in which the components of themethod may be lyophilized and retain bioconjugate synthesis capabilitywhen rehydrated.

Embodiment 19

The method of any of embodiments 15-18 where the goal is on-demandvaccine production.

Embodiment 20

The method of any of embodiments 15-19 where the goal is vaccineproduction in resource-limited settings.

Embodiment 21

A kit for synthesizing a N-glycosylated carrier protein in vitro, thekit comprising one or more of the following components: (i) a firstcomponent comprising a cell lysate that comprises an orthogonaloligosaccharyltransferase (OST); (ii) a second component comprising acell lysate that comprises an O-antigen (e.g., lipid-linkedoligosaccharides (LLOs) comprising O-antigen; (iii) a third componentcomprising a transcription template and optionally a polymerase forsynthesizing an mRNA from the transcription template encoding a carrierprotein, the carrier protein comprising an inserted and/or a naturallyoccurring consensus sequence, N-X-S/T, wherein X may be any natural orunnatural amino acid except proline.

Embodiment 22

The kit of embodiment 21, wherein one or more of the first component,the second component, and the third component are lyophilized and retainbiological activity when rehydrated.

Embodiment 23

The kit of embodiment 21 or 22, wherein the first component cell lysateis produced from a source strain (e.g., E. coli) that overexpresses agene encoding the orthogonal OST (e.g. C. jejuni PglB).

Embodiment 24

The kit of any of embodiments 21-23, wherein the second component celllysate is produced from a source strain that overexpresses a syntheticglycosyltransferase pathway (e.g., the biosynthetic machinery to producethe Franciscella tularensis Schu S4 O-antigen (FtLLOs lysate) or thebiosynthetic machinery to produce the enterotoxigenic E. coli O78lipid-linked oligosaccharides (EcO78LLOs lysate).

Embodiment 25

A method for cell-free production of a glycoprotein which optionally maybe a bioconjugate suitable for use as a vaccine, the method comprising:(a) mixing a first cell lysate comprising an orthogonaloligosaccharyltransferase (OST) and a second cell lysate that comprisesan O-antigen (e.g., as lipid-linked oligosaccharides (LLOs)) to preparea cell-free protein synthesis reaction; (b) transcribing and translatinga carrier protein in the cell-free protein synthesis reaction (e.g.,optionally by adding a transcription template for the carrier proteinand/or a polymerase to the cell-free protein synthesis reaction), thecarrier protein comprising an inserted and/or naturally occurringconsensus sequence, N-X-S/T, wherein X may be any natural or unnaturalamino acid except proline; and (c) glycosylating the carrier protein inthe cell-free protein synthesis reaction with the bacterial O-antigen.

Embodiment 25

The method of embodiment 24, wherein the second cell lysate comprisesthe O-antigen as part of lipid-linked oligosaccharides (LLOs).

Embodiment 26

The method of embodiment 24 or 25, further comprising formulating theglycoprotein as a vaccine composition optionally including an adjuvant.

Embodiment 27

A vaccine prepared by any of the foregoing methods and/or kits.

Embodiment 28

A vaccination method comprising administering the vaccine of embodiment27 to a subject in need thereof.

EXAMPLES

The following Examples are illustrative and are not intended to limitthe scope of the claimed subject matter.

Example 1—Method for Rapid In Vitro Synthesis of Bioconjugate VaccinesVia Recombinant Production of N-Glycosylated Proteins in ProkaryoticCell Lysates

Abstract

Conjugate vaccines are among the safest and most effective methods forprevention of life-threatening bacterial infections [1-10]. Bioconjugatevaccines are a type of conjugate vaccine produced via protein glycancoupling technology (PGCT), in which polysaccharide antigens areconjugated via N-glycosylation to recombinant carrier proteins using abacterial oligosaccharyltransferase (OST) in living Escherichia colicells [11]. Bioconjugate vaccines have the potential to greatly reducethe time and cost required to produce antibacterial vaccines. However,PGCT is limited by i) the length of in vivo process developmenttimelines and ii) the fact that FDA-approved carrier proteins, such asthe toxins from Clostridium tetani and Corynebacterium diptheriae, havenot yet been demonstrated to be compatible with N-linked glycosylationin living E. coli. Here, we have applied cell-free glycoproteinsynthesis (CFGpS) technology to enable rapid in vitro production ofbioconjugate vaccines against pathogenic strains of Escherichia coli andFranscicella tularensis in reactions lasting 20 hours. Due to themodular nature of the CFGpS system, this cell-free strategy could beeasily applied to produce bioconjugates using FDA-approved carrierproteins or additional vaccines against pathogenic bacteria whosesurface antigen gene clusters are known [11] [9] [7, 10] [3, 5, 6, 8].We further show that this system can be lyophilized and retainbioconjugate synthesis capability, demonstrating the potential foron-demand vaccine production and development in resource-poor settings.This work represents the first demonstration of bioconjugate vaccineproduction in E. coli lysates and has promising applications as aportable prototyping or production platform for antibacterial vaccinecandidates.

Background and Significance

Glycosylation, or the attachment of glycans (sugars) to proteins, is themost abundant post-translational modification in nature and plays apivotal role in protein folding and activity [1-4]. When it was firstdiscovered in the 1930s [12], glycosylation was thought to be exclusiveto eukaryotes. However, glycoproteins were also discovered in archaea inthe 1970s [13, 14], and in bacteria in the late 1990s and early 2000s[15, 16], establishing glycosylation as a central post-translationalmodification in all domains of life. A vast diversity of glycanstructures, including both linear and highly branched polysaccharidechains, have been described [17], giving rise to exponentially increasedinformation content compared to other polypeptide modifications [18].

As a consequence of its role in protein structure and informationstorage, glycosylation is involved in a variety of biological processes.In eukaryotes, glycoproteins are involved in immune recognition andresponse, intracellular trafficking, and intercellular signaling[19-22]. Furthermore, changes in glycosylation have been shown tocorrelate with disease states, including cancer [23-25], inflammation[26-29], and Alzheimer's disease [30]. In prokaryotes, glycosylation isknown to play important roles in virulence and host invasion [31-33].Based on the vital role of glycosylation in numerous biologicalprocesses, it has been proposed that the central dogma of biology beadapted to include glycans as a central component [34].

The most common forms of glycosylation are asparagine linked (N-linked)and serine (Ser) or threonine (Thr) linked (O-linked) [35]. N-linkedglycosylation is characterized by the addition of a glycan moiety to theside chain nitrogen of asparagine (Asn) residues by anoligosaccharyltransferase (OST) that recognizes the consensus sequenceAsn-X-Ser/Thr, where X is any amino acid except proline [36, 37]. Thisprocess occurs in the endoplasmic reticulum and aids in protein folding,quality control, and trafficking [38]. O-linked glycosylation occurs inthe Golgi apparatus following the attachment of N-glycans. UnlikeN-linked glycosylation, there is no known consensus sequence forO-linked glycosylation [39, 40]. Despite the importance of glycans inbiology, glycoscience was recently identified as an understudied field.A 2012 National Research Council of the U.S. National Academies reporthighlighted the critical need for transformational advances inglycoscience [41]. The discovery of glycosylation pathways in bacteriais enabling new discoveries about this important post-translationalmodification [42, 43], but new synthetic and analytical tools are neededto advance the field.

Since the recent discovery of bacterial glycosylation, proteins bearingN- and O-linked glycans have been found in a number of bacteria [44,45]. The best-studied bacterial glycosylation system is the pgl pathwayfrom Campylobacter jejuni, which has been shown to express functionallyin Escherichia coli (FIG. 1) [46]. In C. jejuni, proteins areN-glycosylated with the 1.406 kDa GlcGalNAc₅Bac heptasaccharide (Glc:glucose, GalNAc: N-acetylgalactosamine, Bac: bacillosamine) GTs assemblethe heptasaccharide onto the lipid anchor undecaprenol pyrophosphate(Und-PP), which is then used as a substrate for the OST (PglB) forN-linked glycosylation [47-49]. This pathway is significantly simplerthan eukaryotic glycosylation pathways, and has been leveraged toincrease our understanding of the mechanism of N-linked glycosylation[42, 43].

Though not all bacteria synthesize glycoproteins, glycosylation is ofteninvolved in the synthesis of the bacterial cell wall. Lipopolysaccharide(LPS) molecules are a major component of the outer membrane of manyGram-negative bacteria, and are made up of a lipid anchor, anoligosaccharide core, and a variable polysaccharide region known as theO-antigen [32]. Capsular polysaccharides (CPS) are another type ofsurface polysaccharide similar in structure to LPS, except that in thiscase the polysaccharide region is linked directly to lipid A or aphospholipid anchor [31]. LPS O-antigens (O-PS) and CPS are one of themain tools used by bacterial pathogens for survival in hostile hostenvironments and for host invasion [50-53]. As a result, elucidation ofCPS and O-PS biosynthesis mechanisms is of interest for antibiotic andantibacterial vaccine development.

The rise of antibiotic-resistant bacterial strains necessitates thedevelopment of novel strategies for treatment and prevention oflife-threatening bacterial infections. In 2013, the Center for DiseaseControl and Prevention released a report citing antibiotic resistance asone of United States' most serious health threats. Conjugate vaccines,which consist of CPS or O-PS antigens covalently linked to carrierproteins, are among the safest and most effective preventative measuresagainst bacterial infections and have been used to reduce the incidenceof Streptococcus pneumoniae, Neisseria meningitides, and Haemophilusinfluenza infection [1-10]. Because polysaccharide antigens cannotdirectly activate naïve T cells, they must be conjugated to a carrierprotein in order to induce long-lasting immunological memory [54].However, existing technologies for producing conjugate vaccines arecomplex, involve multiple processing and purification steps, and theresulting products are ill-defined (FIG. 2, top) [2]. Additionally,these processes are time-consuming and can require large-scalefermentation of pathogenic bacteria, making conjugate vaccinesprohibitively expensive for vaccination campaigns in developing nations.

The production of recombinant O antigen-protein conjugates in living E.coli cells was recently accomplished using bacterial N-glycosylationmachinery (FIG. 2, bottom) [11]. These so-called bioconjugate vaccineshave the potential to reduce the cost and time required forantibacterial vaccine production. Bioconjugates have been developedagainst several bacterial targets, including Franciscella tularensis[4], Pseudomonas aeruginosa [11], Salmonella enterica [9], Shigelladynsenteriae [7, 10], Shigella flexneri [8], Staphylococcus aureus [5],Brucella abortus [3], and Burkholderia pseudomallei [6]. An in vitromethod for bioconjugate production could shorten process developmenttimelines for novel antibacterial vaccines from months to weeks [55].

Cell-free protein synthesis (CFPS) is an emerging field that allows forthe production of proteins in crude cell lysates [55, 56]. CFPStechnology was first used over 50 years ago by Nirenberg and Matthaei todecipher the genetic code [57]. In the late 1960s and early 1970s, CFPSwas employed to help elucidate the regulatory mechanisms of the E. colilactose [58] and tryptophan [59] operons. In the last two decades, CFPSplatforms have experienced a surge in development to meet the increasingdemand for recombinant protein expression technologies [55].

CFPS offers several advantages for recombinant protein expression. Inparticular, the open reaction environment allows for addition or removalof substrates for protein synthesis, as well as precise, on-linereaction monitoring. Additionally, the CFPS reaction environment can bewholly directed toward and optimized for production of the proteinproduct of interest. CFPS effectively decouples the cell's objectives(growth & reproduction) from the engineer's objectives (proteinoverexpression & simple product purification), which has provenadvantageous for the production of complex proteins and proteinassemblies, including membrane proteins [60-63], bispecific antibodies[64], antibody-drug conjugates [65], and virus-like particle vaccines[66-68]. Overall, CFPS technology allows for shortened protein synthesistimelines and increased flexibility for addition or removal ofsubstrates compared to in vivo approaches. The E. coli CFPS system inparticular has been widely adopted because of i) its high batch yields,with up to 2.3 g/L of green fluorescent protein (GFP) reported [69], ii)inexpensive required substrates [70-72], and iii) the ability tolinearly scale reaction volumes over 10⁶ L [73].

Glycosylation is possible in some eukaryotic CFPS systems, includingICE, CHO extract, and a human leukemia cell line extract [74-77].However, these platforms harness the endogenous machinery to carry outglycosylation, meaning that i) the possible glycan structures arerestricted to those naturally synthesized by the host cells and ii) theglycosylation process is carried out in a “black box” and thus difficultto engineer or control. The development of a highly active E. coli CFPSplatform has prompted recent efforts to enable glycoprotein productionin E. coli lysates through the addition of orthogonal glycosylationcomponents. In one study, Guarino and DeLisa demonstrated the ability toproduce glycoproteins in E. coli CFPS by adding purified lipid-linkedoligosaccharides (LLOs) and the C. jejuni OST to a CFPS reaction. Yieldsof between 50-100 μg/mL of AcrA, a C. jejuni glycoprotein, were achieved[78]. Despite these recent advances, bacterial cell-free glycosylationsystems have been limited by their inability to co-activate efficientprotein synthesis and glycosylation. We recently developed a cell-freeglycoprotein synthesis (CFGpS) system that addresses this limitation byenabling modular, coordinated transcription, translation, andN-glycosylation of proteins in E. coli lysates selectively enriched withglycosylation enzymes (see WO 2017/117539, the content of which isincorporated herein by reference in its entirety). Here, we apply thistechnology platform to the production of bioconjugate vaccines to yielda methodology for rapid, modular in vitro expression of bioconjugates.

Results and Discussion

Cell-free Glycoprotein Synthesis (CFGpS) for Bioconjugate VaccineProduction.

While protein-glycan coupling technology (PGCT) represents a simplifiedand cost-effective strategy for bioconjugate vaccine production, it hasthree main limitations. First, process development timelines,glycosylation pathway design-build-test (DBT) cycles, and bioconjugateproduction are all limited by cellular growth. Second, it has not yetbeen shown whether FDA-approved carrier proteins, such as the toxinsfrom Clostridium tetani and Corynebacterium diptheriae, have not yetbeen demonstrated to be compatible with N-linked glycosylation in livingE. coli. Third, select non-native glycans are known to be transferredwith low efficiency by the C. jejuni OST, PglB [9].

A modular, in vitro platform for production of bioconjugates has thepotential to address all of these limitations. Here, we demonstrate thatbioconjugates against pathogenic strains of Franciscella tualrensis andEscherichia coli can be produced through coordinated in vitrotranscription, translation, and N-glycosylation in cell-freeglycoprotein synthesis (CFGpS) reactions lasting just 20 hours. Thissystem has the potential to reduce process development and distributiontimelines for novel antibacterial vaccines from weeks to days. Further,because of the modular nature of the CFGpS platform and the fact thatcell-free systems have demonstrated advantages for production ofmembrane proteins compared to living cells [60-63], this method could bereadily applied to produce bioconjugates using FDA-approved carrierproteins, such as the Clostridium tetani and Corynebacterium diptheriaetoxins, which are membrane localized. This could be accomplished simplyby supplying plasmid encoding these carrier proteins to CFGpS reactions.Additionally, because of the modular nature of CFGpS, the in vitroapproach could be used to prototype other natural or engineered homologsof the archetypal C. jejuni OST, such as those described recently [9,79, 80], to identify candidate OSTs with improved efficiency fortransfer of O-antigen LLOs of interest. This can be accomplished byenriching lysates with OSTs of interest and mixing them with LLO lysatesin mixed lysate CFGpS reactions, as we have described previously (Jewettlab, unpublished data; U.S. Provisional Patent Application 62/273,124).Finally, we demonstrate that cell-free bioconjugate synthesis reactionscan be lyophilized and retain bioconjugate synthesis capability,demonstrating the potential of the CFGpS system for on-demand, portable,and low cost production or development efforts for novel vaccines. Thisnovel method for in vitro bioconjugate vaccine production hasdemonstrated advantages for rapid, modular, and portable vaccineprototyping and production compared to existing methods.

Bioconjugate Vaccines Against F. tularensis can be Synthesized In VitroVia CFGpS.

Franciscella tularensis is a gram-negative bacterium that causestularemia. F. tularensis is considered one of the most infectiousbacteria known to man: a single inhaled bacillus results in infection of40-50% of individuals, with a mortality rate of up to 30% [81]. The genelocus for O-antigen biosynthesis was recently characterized [82] andused to create a bioconjugate vaccine in living E. coli cells [4]. Wedecided to use this pathway to produce an anti-F. tularensisbioconjugate vaccine using CFGpS. We hypothesized that anti-F.tularensis bioconjugate vaccines can be synthesized in vitro using CFGpStechnology (FIG. 3).

We hypothesized that Und-PP-linked polysaccharide antigens can beenriched in S30 lysates via overexpression of orthogonal glycosylationmachinery in an E. coli CFGpS chassis strain lacking the WaaL O-antigenligase. To test this hypothesis, we produced lysates from CLM24 cellsoverexpressing the C. jejuni OST, PglB (CjOST lysate) and thebiosynthetic machinery to produce the Franciscella tularensis Schu S4 Oantigen (FtLLO lysate). This 17 kb gene cluster from F. tularensis SchuS4 contains 15 predicted open reading frames (ORFs) and producesoligosaccharides comprised of the repeating unit GalNAcAN₂QuiNAcQui4NFm(GalNAcAN: 2-acetamido-2-deoxy-D-galacturonamide, QuiNAc:2-acetamido-2,6-dideoxy-D-glucose, Qui4NFm:4,6-dideoxy-4-formamido-D-glucose) (FIG. 6A) [82]. Lysates were preparedvia high-pressure homogenization, to encourage formation of solubleinverted membrane vesicles, which carry membrane-bound components, suchas FtLLOs and CjOST, into the crude lysate.

We hypothesized that anti-F. tularensis bioconjugate vaccines can besynthesized in vitro using CFGpS technology. To produce bioconjugatesbearing the F. tularensis O polysaccharide (FtO-PS), CjPglB lysate wasmixed with FtLLO lysate in CFGpS reactions lasting 20 hours at 30° C.These reactions contained plasmid encoding either i) super-folder greenfluorescent protein engineered to include a DQNAT sequon in a flexiblelinker inserted at residue T216 and a C-terminal His tag(sfGFP-21-DQNAT-6×His) or ii) an engineered maltose binding proteinconstruct with four C-terminal repeats of the DQNAT sequon and aC-terminal His tag (MBP-4×DQNAT-6×His). Bioconjugates composed of E.coli O-antigens conjugated to MBP-4×DQNAT-6×His were recently shown toelicit humoral and cellular immunity in mice [83]. Within the first hourof the CFGpS reaction, reaction mixtures were spiked with manganesechloride (MnCl₂) and n-dodecyl-β-D-maltopyranoside (DDM) detergent atfinal concentrations of 25 mM MnCl₂, 0.1% w/v DDM to optimize CjPglBactivity (Jewett lab, unpublished data). After the CFGpS reactions wereallowed to proceed for a total of 20 hours at 30° C., glycosylationactivity was assayed via immunoblotting with anti-His antibody and acommercial monoclonal antibody specific to FtO-PS. The ladder-likepattern observed when the FtLLO and CjPglB lysates are mixed isindicative of FtLLO attachment and results from O-PS chain lengthvariability through the action of the Wzy polymerase (FIG. 4A) [4, 11].These results demonstrate the integration of coordinated cell-freecarrier protein synthesis and O-antigen attachment for the first time.Furthermore, we show that cell-free bioconjugate synthesis occurs injust 20 hours, making this cell-free platform an attractive option forrapid production and prototyping of bioconjugate vaccine candidates.

Bioconjugate Vaccines Against E. coli O78 and Other Pathogenic Strainscan be Readily Synthesized Due to the Modular Nature of CFGpS Platform.

To demonstrate the generality and modularity of the CFGpS approach forbioconjugate production, we sought to produce bioconjugates against anadditional pathogenic bacterium. We decided to synthesize bioconjugatesagainst enterotoxigenic E. coli strain O78, since its biosyntheticpathway has been described previously [84] and there exists a commercialantiserum specific to this strain. The E. coli O78 gene cluster wascloned and is encoded in plasmid pMW07-O78. Expression of the E. coliO78 gene cluster results in production of an O-antigen with therepeating unit GlcNAc₂Man₂ (GlcNAC: N-acetylglucosamine; Man: mannose)(FIG. 6B)

We prepared lysates enriched with E. coli O78 antigen LLOs (EcO78LLOlysate) from CLM24 cells expressing pMW07-O78 using the same methoddescribed above. We then mixed the EcO78LLO lysate with CjOST lysate inCFGpS reactions containing plasmid encoding either sfGFP21-DQNAT-6×Hisor MBP-4×DQNAT-6×His. CFGpS reactions were run as described above.Glycosylation activity was assayed via immunoblotting with an anti-Hisantibody and a commercial antiserum against the E. coli O78 strain.Again, a ladder-like pattern is observed when both EcO78LLO lysate andCjOST lysates are present in the reaction, indicating successfulconjugation of the EcO78-PS (FIG. 4B). The bioconjugates were alsocross-reactive with the anti-EcO78 serum (data not shown). These resultsdemonstrate the modular nature of the in vitro approach, and provideevidence that the CFGpS platform could be used to produce bioconjugatevaccines targeting many different pathogenic bacterial strains.Attractive candidates include pathogenic bacteria whose surface antigengene clusters are known, including Pseudomonas aeruginosa [11],Salmonella enterica [9], Shigella dynsenteriae [7, 10], Shigellaflexneri [8], Staphylococcus aureus [5], Brucella abortus [3], andBurkholderia pseudomallei [6]. These results demonstrate the potentialof the in vitro system for rapid production or prototyping of vaccinecandidates directed against diverse pathogenic bacteria. Moreover,because synthesis can be achieved in just 20 h with different O-PSantigens, this cell-free platform represents an attractive option forrapid production and prototyping of novel bioconjugate vaccinecandidates.

Lyophilized CFGpS Reactions Retain Bioconjugate Synthesis Capability.

A major limitation of traditional conjugate vaccines is their high cost,due to their time consuming production process. By comparison,bioconjugate vaccines are less expensive to produce, however, productiontime is dictated by the time required to ferment and purify proteinsfrom living cells. We wanted to test whether our CFGpS reactions couldbe freeze-dried for potential room-temperature storage and distributionto enable faster production and broader-reaching vaccination campaignsand development efforts compared to in vivo approaches. To test whetherthis was possible, CFGpS reactions were prepared containing either CjOSTlysate or FtLLO lysate alone, or a mixture of both CjOST and FtLLOlysates. These reactions were then lyophilized and reconstituted with 15μL nuclease-free water. The reactions contained plasmid encoding eithera short chain antibody fragment with a C-termnal DQNAT sequon followedby a His tag (scFv13-R4-DQNAT-6×His) or MBP-4×DQNAT-6×His. Pre-mixedreactions (lanes 1, 2, 5, 6) were run directly following reconstitution,while reactions containing CjOST lysate or FtLLO lysate alone were mixedfollowing reconstitution (lanes 3, 4, 7). The FtO-PS is attached to thetarget protein when the DQNAT sequon is synthesized and both CjOSTlysate or FtLLO lysate are present in the reaction (lanes 2, 4, 6, 7).These results confirm that the CFGpS reaction mixture can be lyophilizedwithout loss of bioconjugate synthesis capability, highlighting thepotential of our technology for portable, on-demand vaccine productionand long-term, refrigeration-free storage.

CONCLUSIONS

We describe here a novel method for coordinated in vitro transcription,translation, and conjugation of vaccine antigens to carrier proteins.This in vitro approach uniquely (i) decouples cell viability fromglycosylation activity and enables reduction of cellular metabolicburden through in vitro reconstitution of glycosylation components, (ii)permits design-build-test (DBT) iterations on individual glycosylationcomponents, and (iii) allows for assembly of glycosylation pathwayswithin well-defined experimental conditions including chemical andphysical manipulations not possible in cells. Further, the system hasobvious and commercially attractive applications to producingbioconjugates using FDA-approved carrier proteins, such as the toxinsfrom Clostridium tetani and Corynebacterium diptheriae. This novelmethod for in vitro bioconjugate vaccine production has demonstratedadvantages for rapid, modular, and portable vaccine prototyping andproduction compared to existing methods.

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In the foregoing description, it will be readily apparent to one skilledin the art that varying substitutions and modifications may be made tothe invention disclosed herein without departing from the scope andspirit of the invention. The invention illustratively described hereinsuitably may be practiced in the absence of any element or elements,limitation or limitations which is not specifically disclosed herein.The terms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention that in theuse of such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, but it is recognizedthat various modifications are possible within the scope of theinvention. Thus, it should be understood that although the presentinvention has been illustrated by specific embodiments and optionalfeatures, modification and/or variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention.

Citations to a number of patent and non-patent references are madeherein. The cited references are incorporated by reference herein intheir entireties. In the event that there is an inconsistency between adefinition of a term in the specification as compared to a definition ofthe term in a cited reference, the term should be interpreted based onthe definition in the specification.

We claim:
 1. A method for synthesizing an N-glycosylated carrier proteinfor a polysaccharide via coordinated transcription, translation, andN-glycosylation in vitro, the method comprising: (i) transcribing andtranslating a carrier protein in a cell-free protein synthesis reaction,the carrier protein comprising an inserted consensus sequence, N-X-S/T,wherein X may be any natural or unnatural amino acid except proline;(ii) glycosylating the carrier protein in the cell-free proteinsynthesis reaction with at least one polysaccharide, wherein the atleast one polysaccharide is at least one bacterial O-antigen.
 2. Themethod of claim 1, wherein the bacterial O-antigen is from E. coli. 3.The method of claim 1, wherein the bacterial O-antigen is fromFranciscella tularensis.
 4. The method of claim 1, further comprisingformulating the N-glycosylated carrier protein as a vaccine compositioncomprising the N-glycosylated carrier protein.
 5. The method of claim 1,wherein the vaccine composition further comprises an adjuvant.
 6. Themethod of claim 1 wherein the carrier protein is an engineered variantof E. coli maltose binding protein (MBP).
 7. The method of claim 1wherein the carrier protein is a detoxified variant of the toxin fromClostridium tetani.
 8. The method of claim 1 wherein the carrier proteinis a detoxified variant of the toxin from Corynebacterium diptheriae. 9.The method of claim 1, wherein the glycosylating step utilizes anoligosaccharyltransferase (OST) which is a naturally occurring bacterialhomolog of C. jejuni PglB.
 10. The method of claim 1, wherein theglycosylating step utilizes an OST that is an engineered variant of C.jejuni PglB.
 11. The method of claim 1, wherein the wherein theglycosylating step utilizes an OST that is a naturally occurringarchaeal OST.
 12. The method of claim 1 wherein the wherein theglycosylating step utilizes an OST which is a naturally occurringsingle-subunit eukaryotic OST.
 13. A kit for synthesizing aN-glycosylated carrier protein in vitro, the kit comprising: (i) a firstcomponent comprising a cell lysate that comprises an orthogonaloligosaccharyltransferase (OST); (ii) a second component comprising acell lysate that comprises an O-antigen; (iii) a third componentcomprising a transcription template and a polymerase for synthesizing anmRNA from the transcription template encoding a carrier protein, thecarrier protein comprising an inserted consensus sequence, N-X-S/T,wherein X may be any natural or unnatural amino acid except proline 14.The kit of claim 13, wherein one or more of the first component, thesecond component, and the third component are lyophilized.
 15. The kitof claim 13, wherein the first component cell lysate is produced from asource strain that overexpresses a gene encoding the orthogonal OST. 16.The kit of claim 13, wherein the second component cell lysate isproduced from a source strain that overexpresses a syntheticglycosyltransferase pathway.
 17. The kit of claim 16, wherein the secondcomponent cell lysate comprises O-antigen from F. tularensis Schu S4lipid-linked oligosaccharides (FtLLOs) or O-antigens fromenterotoxigenic E. coli O78 lipid-linked oligosaccharides (EcO78LLOs).18. A method for cell-free production of a glycoprotein, the methodcomprising: (a) mixing a first cell lysate comprising an orthogonaloligosaccharyltransferase (OST) and a second cell lysate that comprisesan O-antigen to prepare a cell-free protein synthesis reaction; (b)transcribing and translating a carrier protein in the cell-free proteinsynthesis reaction, the carrier protein comprising an inserted consensussequence, N-X-S/T, wherein X may be any natural or unnatural amino acidexcept proline; and (c) glycosylating the carrier protein in thecell-free protein synthesis reaction with the bacterial O-antigen. 19.The method of claim 18, wherein the second cell lysate comprises theO-antigen as part of lipid-linked oligosaccharides (LLOs).
 20. Themethod of claim 18, further comprising formulating the glycoprotein as avaccine composition.